Chromosome analysis in pachytene has been considered a powerful tool in cytogenetic and evolutionary studies carried out in several plant species. Many morphological details of chromosomes can be observed during this stage, although overlapping of chromosomes may hinder the analysis. Considering this limitation, a new cytogenetic methodology was developed for maize meiotic chromosomes. This method consists of submitting the pollen mother cells (PMCs) to an enzymatic solution, which promotes PMC release from the anthers and subsequent digestion of the cell wall. The bivalents are mechanically released into the cytoplasm and spread out over the slide. In this slide preparation the chromosomes were isolated and spread over in the same plane of focus. The preservation of morphological traits of the bivalents, and the near complete absence of background or artifacts, resulted in high quality preparations, adequate for cytogenetic analysis. The obtainance of individual chromosomes allowed for the detailed analysis of the 10 maize bivalents. This analysis revealed additional data on the morphology and longitudinal differentiations of the chromosomes, facilitating their identification and morphological characterization.